PROJECT SUMMARY Work from many laboratories has established that DNA sensors ? both membrane-localized Toll-like receptors (e.g. TLR9) and intracellular DNA sensors (e.g. cGAS and IFI16) ? sense herpesviral infection. In striking contrast, our knowledge about the relevance of cytoplasmic RNA sensors of the RIG-I-like receptor (RLR) family in the detection of herpesviruses remains elusive. Furthermore, the physiological RNA molecules that are recognized by RIG-I and the related sensor MDA5 during herpesvirus infection are currently unknown. The proposed study builds on a recent discovery by the Gack laboratory that RIG-I and the intracellular DNA sensors cGAS and IFI16 sense herpes simplex virus 1 (HSV-1) infection in a temporally distinct manner. Using next-generation RNA sequencing (RNA-seq), we identified that a subset of cellular non-coding RNAs, rather than viral RNAs, are bound to and activate RIG-I during HSV-1 infection. Molecular and cell biological studies in HSV-1-infected human cells revealed that viral perturbation of the natural life cycle of these host-derived RNAs allows their recognition by the sensor RIG-I, thereby stimulating the innate immune response. Using a coordinated series of molecular, biochemical and cell biological approaches combined with RNA-seq technology in HSV-1-infected cells, we will identify and characterize in precise detail the physiological RNA ligands recognized by RIG-I and MDA5 during HSV-1 infection (Aim 1). Furthermore, we will define the molecular details of how viral perturbation of the life cycle of immunostimulatory RNAs triggers an RLR-mediated immune response (Aim 2). Our studies will provide a molecular understanding of RLR-mediated innate sensing of herpesviral infection, which may provide the foundation for new therapeutic approaches or help design better vaccines.